Screening Of High-yield β-Carotene Mutant Strains By Ions Implantation And Cell Fixation Technology Of Dunaliella Salina

Dunaliella salina(Chlorophyta,Chlorophyceae) is a unicellular wall-less biflagellate alga,coated by Glycoprotein and Neuraminic acid.It content plenty of glycerol,carotene, protein and multi-vitamine,so it is worthy of economy and research.In this paper a system investigation had been done on the ions implantation factors of Dunaliella salina and effect to the cell growth andβ-carotene accumulation of the mutant strains.A system study also had been done on the alga fixation technology in terms of microbeam radiation conditions. The results as follows:1.By Ar⁺ ions implantation with different doses of 10keV energy,we studied the cell survival rate and mutation rate and confirmed that the optimum Ar⁺ ions implantation dose is 1.04×10¹⁵ Ar⁺/cm².2.A high-yieldβ-carotene mutant strain named CH2 was obtained after few rounds of screening,whoseβ-carotene total content is 15.075mg/L and single cellβ-carotene content is 2.258pg/cell,which has increased 42%in contrast to the original strain. After transferred generation experiments,it is finally indicated that this algae had the heredity stability in the synthesisβ-carotene character.3.This paper has studied changes of cell growth and pigment accumulation bewteen the original strain and the mutant strains.The result indicated that after ion implantation the growth curves of the positive mutant strains have obvious rise to the original algae,the growth speed increases,β-carotene total content increases from original 8.598mg/L to 14.397mg/L above and the single cellβ-carotene content increases from original 1.59pg/cell to 1.996pg/cell above;The ratio bewteen chlorophyll content andβ-carotene content drops from 2.837mg/L to 2.088mg/L,it is explained that the algae'sβ-carotene accumulation process and cell's growth are being related before its entering the stabilization period,the growth of the mutant stains still have the substantial growing space after entering the stabilization period,β-carotene is accumulating unceasingly;although the chlorophyll content is increasing,the growth rate tends to be slow.4.A technology systerm on Dunaliella salina's cell fixation has been estiblished,the result list as:the optimum cell fixed period is the 6-7th day after the algae's transfer,the NaCl content which most suitable laboratory cell growth is 30g/L;Cell fixing agent Poly-L-Lysine(PLL) density is 1.25mg/mL,the dosage is 1ml,the fixed area diameter is 2mm,;the most suitable staining density and dosage of fluorescein diacetate(FDA) and propidium iodide(PI) double staining are:The FDA density is 5mg/mL,the dosage is the 400ul,PI density is 400ug/L,the dosage is 210uL;The trypsin elution's most suitable elution time is 3～4min and elution dosage 240～250uL,plusing 60rpm raises 12h to be possible to elute the fixed cells completely.In conclusion,the founded Ar⁺ ions implantation technology of Dunaliella salina,the gotten high-yieldβ-carotene mutant strain and the established single cell fixation technology have laid the theoretical and technical foundation for the character improvement and single cell orientable radialization of Dunaliella salina.